Glucose-derived glycolytic ATP is a vital cellular fuel during hypoxic inhibition of oxidative metabolism. Some cells respond acutely to metabolic stress by activating a specific cell-surface glucose transport protein - GLUT1. The broad goal of this proposal is to understand how GLUT1 is activated and why only some cells respond in this way. Our efforts focus on 3 questions. 1) What are the regulated steps of the transport cycle? 2) What cellular signals modulate these transport steps? 3) How do these signals modulate the regulated steps? We address these questions to cells in which GLUT1-mediated sugar transport is regulated (Clone 9 cells, K562 cells, rat cardiomyocytes and human and avian red cells) and will contrast these findings with those obtained in CHO cells which lack regulated transport. Specific Aim 1 uses net and exchange sugar transport measurements to test the hypothesis that GLUT1 functions as an antiporter in all regulated cells under basal conditions, and as a uniporter in stimulated cells. These studies will also show which transport steps are stimulated during GLUT1 activation. Specific Aim 2 uses GLUT1 reconstitutions to test whether ATP/AMP regulation of human red cell glucose transport is mediated by direct nucleotide/GLUT1 interaction and if so, identifies which transport steps are affected. Specific Aim 3 asks whether increased AMP-activated protein kinase (AMPK) activities seen in metabolically inhibited avian red cells also occur in other regulated cells. If so, we will use AMPK agonists to determine whether this activation mediates GLUT1 stimulation. Using MAP kinase inhibitors, Specific Aim 4 asks whether acute (protein synthesis inhibition independent) activation of GLUT1 by anisomycin is mediated by activation of the anisomycin-sensitive MAP kinases. If so, we will determine which MAPK subfamily (JNK or p38) mediates this response and whether this activation is upstream, downstream or in parallel to factors mediating transport stimulations during cellular metabolic depletion. Specific Aim 5 asks whether AMP/ATP interactions with GLUT1 occur at a single nucleotide binding site by competition analysis of nucleotide binding to GLUT1. Com-ponents of this nucleotide binding pocket(s) will be identified using a photoaffinity labelling/peptide mapping/N-terminal sequencing strategy. Specific Aim 6 tests the hypothesis that the newly described, ATP-sensitive intracellular sugar binding complex of erythrocytes is in fact GLUT1. If not we describe a purification strategy to identify this complex. This sugar binding complex may account for membrane compartmentation of glucose-derived glycolytic ATP in some cells.